Anti fibrinogen

Platelet count and parameters were determined using an automated hematology analyzer (Sysmex, Kobe, Japan). Platelet reactivity was determined in citrated whole blood (3.2% sodium citrate, Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometry based assay as previously described between 1 and 3 h after blood collection^{52 (link)}. Platelets were ex vivo stimulated with ADP (1.2 and 125 μM; Sigma-Aldrich, Zwijndrecht, The Netherlands) and CRP-XL (a kind gift from Prof. Farndale, Cambridge, UK) for 20 min at room temperature. Platelets were stained using anti-CD61 (Beckman Coulter, Brea. CA, USA), anti-P-selectin (Biolegend, San Diego, CA, USA) and anti-fibrinogen (DAKO, Santa Clara, CA) antibodies and fixated in 0.2% paraformaldehyde. Platelets were identified based on Size (FSC), granularity (SSC) and their expression of CD61. Degranulation was determined as the membrane expression of α-granule protein P-selectin and platelet aggregation was quantified as the amount of fibrinogen binding to the activated integrin αIIbβ3. Platelet reactivity was measured on a FC500 flow cytometer (Beckman Coulter, Brea, USA). Data were extracted using Kaluza 2.1 (Beckman Coulter), normalized against quality controls to ensure measurement stability and are expressed as median fluorescence intensity (MFI). A gating strategy is provided in Supplemental Fig. S1.

IgG and albumin extravasation. Brains were cut serially in 20-μm cryosections. Four sections per brain, two at the striatum (0.26 mm and 0.92 mm posterior to the bregma) and another two at the dorsal hippocampus (3.14 mm and 4.16 mm posterior to the bregma) according to a rat brain atlas, were selected for staining. The brain sections were incubated with primary anti-IgG antibody (HRP-conjugated 1:200; AP136P, Chemicon) or anti-albumin antibody (1:200, Genetex) for 2 h at room temperature. Biotin-peroxidase signals were detected using 0.5 mg/mL 3′3′-diaminobenzidine (DAB)/0.003% H₂O₂ as a substrate and were microscopically recorded (Eclipse E400, Nikon). The integrated optic density of IgG and albumin signals were further analyzed and the averaged value at three visual fields in the cortex per sections at ×200 magnification (0.145 mm²/per visual field).
Fibrin leakage. Brain cryosections were probed with primary antibodies, anti-fibrinogen (1:200 Dako) and anti-RECA antiboties (1:100 Abcam) as described in the previous immunefluorescence method.

The following antibodies were utilised in our study: 6E10 (against Aβ1–16) from Covance; MOAB-2 clone 6C3 against Aβ from Merck-Millipore (Burlington, MA, USA); anti-apolipoprotein E (ApoE) and Aquaporin-4 (AQP-4) from Santa Cruz; anti-insulin-degrading enzyme (IDE) and anti-β-actin from Abcam; anti-ionized calcium-binding adaptor molecule 1 (Iba1) from Wako; anti-CD68 from Biolegend; Rat anti-GFAP (clone 2.2B10) from Invitrogen or from DAKO; anti-fibrinogen from DAKO and anti-CD31 from BD Biosciences. All other reagents were purchased from Invitrogen or Sigma, unless otherwise indicated.